Hepatitis E Virus (HEV) Infection in Ireland.

Hepatitis E virus (HEV) is a single stranded RNA virus causing infection worldwide. In developing countries HEV genotypes 1 and 2 spread faeco-orally via water. Recently, infections with HEV have been detected in Europe and North America in patients with no travel history. These are food-borne HEV genotypes 3 and 4, a pig-associated zoonosis. Most infections are asymptomatic but morbidity and chronic infection may occur with prior liver disease or immunosuppression. International seroprevalence rates vary and with improved diagnostics have increased. To determine the current prevalence in this region we studied anonymised serum samples submitted in 2015 for routine testing. We detected anti-HEV IgG in 16/198 (8%) individuals, highest rate in 40-59 year olds (43.8%). This is higher than reported for the same region in 1995 (0.4%) using a previous generation assay. This study provides evidence of HEV circulation in Ireland and reinforces the need for ongoing surveillance.

Visual access to an outdoor range early in life, but not environmental complexity, increases meat chicken ranging behavior.

Not all chickens access an outdoor range when the opportunity is provided. This may be related to the abrupt change in environments from the stable rearing conditions to the complexity of the outdoor range. We aimed to prepare chickens to range by increasing the complexity of the indoor environment early in life with the intention to encourage range use. Mixed sex Cobb500 chickens were allocated to 1 of 3 treatment groups: visual access (VA) treatment provided VA to the outdoor range from day old via transparent pop-hole covers; environmental complexity (EC) treatment provided an artificial haybale, fan with streamers and a solid vertical barrier; Control treatment was a representative conventional environment. Chickens were given access to the outdoor range at 21 d of age. Behavior in the home pen was assessed in wk 1, 2 and 5 and individual ranging behavior was monitored through radio frequency identification (RFID) technology. The VA chickens were more active compared to EC (P = 0.006) and Control (P = 0.007) chickens and spent more time foraging than control chickens (P = 0.036) during the first week of life. More VA chickens accessed the range area compared to EC chickens (P = 0.015). VA chickens accessed the range sooner after they were first provided access and spent more time on the range than EC and control chickens (P < 0.001). Mortality was lower in the VA treatment compared to EC (P = 0.024) and control group (P = 0.002). There was evidence that VA chickens weighed less than Control and EC chickens, however results were inconsistent between age and sex. Hence, providing meat chickens with VA to an outdoor range early in life increased activity in early life, decreased latency to first access the range and increased time on the range and lowered mortality. Future work should aim to understand the mechanism behind these changes in behavior to develop recommendations for producers to implement in commercial conditions.

Desensitization regimens for drug allergy: state of the art in the 21st century.

Adverse reactions to drugs are increasingly being recognized as important contributions to disease in their own right as well as impediments to the best treatment of various conditions, including infectious, autoimmune, and neoplastic maladies. Rapid drug desensitization (RDD) is an effective mechanism for safely administering important medications while minimizing or entirely circumventing such adverse reactions in sensitized patients. We reviewed the literature on RDD in the last 10 years, including our experience from the Brigham and Women's Hospital Desensitization Program with hundreds of patients desensitized to a broad variety of drugs. RDD in our programme has been uniformly successful in patients with hypersensitivity reactions to antibiotics, chemotherapeutics, and monoclonal antibodies. Any reactions that occur during desensitization are generally much less severe than the initial hypersensitivity reaction to the drug, and patients have received the full dose of the desired medication 99.9% of the time out of (796) desensitizations. To date, there have been no fatalities. RDD is a safe and highly effective method for treating sensitized patients with the optimal pharmacologic agents. Its use should be expanded, but because patient safety is paramount, protocols must be created, reviewed, and overseen by allergist-immunologists with special training and experience in modern techniques of desensitization.

Impact of Age, Frailty, and Dementia on Prescribing for Type 2 Diabetes at Hospital Discharge 2012-2016.

BACKGROUND: The risks of intensive blood glucose lowering may outweigh the benefits in vulnerable older people. OBJECTIVES: Our primary aim was to determine whether age, frailty, or dementia predict discharge treatment types for patients with type 2 diabetes (T2D) and related complications. Secondly, we aimed to determine the association between prior hypoglycemia and discharge treatment types. DESIGN, SETTING AND PARTICIPANTS: We conducted a cohort study involving 3,067 patients aged 65-99 years with T2D and related complications, discharged from Melbourne's Eastern Health Hospital Network between 2012 and 2016. MEASUREMENTS: Multinomial logistic regression was used to estimate odds ratios (ORs) with 95% confidence intervals (CI) for the association between age, frailty, dementia and hypoglycemia, and being prescribed insulin-only, non-insulin glucose-lowering drugs (GLDs) or combined insulin and non-insulin GLDs compared to no GLD. International Classification of Diseases-10 codes were used to identify dementia status and prior hypoglycemia; frailty was quantified using the Hospital Frailty Risk Score. RESULTS: Insulin-only, non-insulin GLDs, combined insulin and non-insulin GLDs, and no GLDs were prescribed to 19%, 39%, 20%, and 23% of patients, respectively. Patients >80 years were less likely than patients aged 65-80 to be prescribed any of the GLD therapies, (eg. non-insulin GLDs [OR 0.67; 95%CI 0.55-0.82]), compared to no GLD. Similarly, high vs. low frailty scores were associated with not being prescribed any of the three GLD therapies, (eg. non-insulin GLDs [OR 0.63; 95%CI 0.45-0.87]). However, dementia was not associated with discharge prescribing of GLD therapies. Patients with a hypoglycemia-related admission were more likely than those not hospitalized with hypoglycemia to receive insulin-only (OR 4.28; 95%CI 2.89-6.31). CONCLUSIONS: Clinicians consider age and frailty when tailoring diabetes treatment regimens for patients discharged from hospital with T2D and related complications. There is scope to optimize prescribing for patients with dementia and for those admitted with hypoglycemia.

Effectiveness of interferon-free therapy for the treatment of HCV-patients with compensated cirrhosis treated through the Irish early access program.

BACKGROUND: We investigated the real-world effectiveness of interferon-free regimens for the treatment of patients with compensated cirrhosis infected with hepatitis C virus (HCV). METHOD: Using the Irish national HCV treatment registry, the effectiveness and safety of interferon-free regimens for HCV-infected patients treated between April 2015 and August 2016, was determined. RESULTS: A SVR12 was achieved in 86% of subjects treated with sofosbuvir/ledipasvir ± ribavirin (SOF/LDV±RBV), 93% treated with paritaprevir, ombitasvir and ritonavir combined with dasabuvir ± ribavirin (3D±RBV) and 89% treated with sofosbuvir/daclatasvir ± ribavirin (SOF/DCV±RBV). The discontinuation rate was 5% and the on-treatment mortality rate was 1%. CONCLUSION: The availability of interferon-free regimens represents a significant breakthrough for the treatment of HCV infection. Treatments options, with high SVR12 rates, are now available for patients with compensated cirrhosis who were unsuitable for treatment with interferon-based regimens. Data obtained from studies conducted in real world practice provide robust information fundamental for input into future economic evaluations for agents used for the treatment of HCV infection.

A strategy for obtaining near full-length HCV cDNA clones (assemblicons) by assembly PCR.

Hepatitis C virus (HCV) genotype is a predictor of response, and guides the duration of antiviral therapy. However, with the exception of HCV genotype 1a, 1b and 2a, a limited number of clones from other genotypes exist. Here we report the optimization of long RT-PCR to generate three overlapping amplicons that span the near full length HCV genome from a panel of HCV genotypes (1a, 1b, 2a, 2b, 3a, 4a, 5a). Assembly-PCR (As-PCR) was used to construct near full-length cDNA clones (assemblicons) for each genotype. The optimization of the long RT-PCR on genotype 1a and 1b indicated that QIAamp Viral RNA kit (Qiagen, UK), Expand RT and Expand Long Template PCR system (Roche, UK), were the most efficient in producing the requisite three overlapping amplicons and assemblicons for each genotype. The genotype of each assemblicon was confirmed. Assemblicon generation was only possible when the overlapping amplicons were biotinylated. As-PCR obviated the need for time consuming ligations and cloning. The use of three overlapping amplicons in the construction of HCV assemblicons minimised the chimeric nature of the resultant clone. As-PCR may prove a methodological avenue through which a larger panel of consensus HCV clones could be made available for HCV in vitro investigation.

Strategy for the maximization of clinically relevant information from hepatitis C virus, RT-PCR quantification.

BACKGROUND: The increasing clinical application of viral load assays for monitoring viral infections has been an incentive for the development of standardized tests for the hepatitis C virus. OBJECTIVE: To develop a simple model for the prediction of baseline viral load in individuals infected with the hepatitis C virus. METHODOLOGY: Viral load quantification of each patient's first sample was assessed by RT-PCR-ELISA using the Roche MONITOR assay in triplicate. Genotype of the infecting virus was identified by reverse line probe hybridization, using amplicons resulting from the qualitative HCV Roche AMPLICOR assay. RESULTS: Retrospective evaluation of first quantitative values suggested that 82.4% (n=168/204) of individuals had a viral load between 4.3 and 6.7 log(10) viral copies per ml. A few patients (3.4%; n=7/204) have a serum viremia less than the lower limit of the linear range of the RT-PCR assay. Subsequent, prospective evaluation of hepatitis C viral load of all new patients using a model based on the dynamic range of viral load in the retrospective group correctly predicted the dynamic range in 75.9% (n=33/54). CONCLUSION: The dynamic range of hepatitis C viremia extends beyond the linear range of the Roche MONITOR assay. Accurate determination of serum viremia is substantially improved by dilution of specimens prior to quantification.

Pregnancy and pregnancy outcome in hepatitis C type 1b.

A large cohort of rhesus-negative women in Ireland were inadvertently infected with hepatitis C virus following exposure to contaminated anti-D immunoglobulin in 1977-8. This major iatrogenic episode was discovered in 1994. We studied 36 women who had been infected after their first pregnancy, and compared them to an age- and parity-matched control group of rhesus-positive women. The presence of hepatitis C antibody was confirmed in all 36 by enzyme-linked immunosorbent assay and by recombinant immunoblot assay, while 26 (72%) of the cohort were HCV-RNA-positive (type 1b) on PCR testing. In the 20 years post-infection, all members of the study group had at least one pregnancy, and mean parity was 3.5. They had a total of 100 pregnancies and 85 of these went to term. There were four premature births, one being a twin pregnancy, and 11 spontaneous miscarriages. One miscarriage occurred in the pregnancy following HCV infection. There were two neonatal deaths due to severe congenital abnormalities in the PCR-positive women. Of the children born to HCV-RNA positive mothers, only one (2.3%) tested positive for the virus. Significant portal fibrosis on liver biopsy was confined to HCV-RNA-positive mothers apart from one single exception in the antibody-positive HCV-RNA-negative group. Comparison with the control group showed no increase in spontaneous miscarriage rate, and no significant difference in obstetric complications; birth weights were similar for the two groups.

Deoxyribonucleic acid hybridization method for the detection of Listeria in dairy products, seafoods, and meats: collaborative study.

The method is based on the hybridization of synthetic deoxyribonucleic acid probes to ribosomal ribonucleic acid sequences unique to Listeria. This method was compared to 2 culture methods: the U.S. Food and Drug Administration method for the detection of Listeria in dairy products and seafoods and the U.S. Department of Agriculture, Food Safety and Inspection Service method for Listeria in meats. Six food types with replicate samples containing various concentrations of Listeria were analyzed by the collaborating laboratories. Listeria was detected in 774 samples using the DNAH method and in 772 samples using a culture method. The DNAH and culture methods were in agreement for 668 samples containing Listeria and 306 samples without Listeria. The overall rate of agreement between methods was 82.3%. The method has been adopted first action by AOAC INTERNATIONAL.

Prospective study of one- vs two-unit umbilical cord blood transplantation following reduced intensity conditioning in adults with hematological malignancies.

As the threshold nucleated cell dose for one-unit umbilical cord blood (UCB) in adults has not to date been firmly established, we prospectively compared one- vs two-unit UCB transplantation after reduced intensity conditioning (RIC) in adult patients with hematological malignancies. Study design specified one-UCB unit if the cryopreserved total nucleated cell (TNC) dose was 2.5 × 10(7)/kg recipient weight, otherwise two units matched at minima of 4/6 HLA loci to the patient and 3/6 to each other were infused. A total of 27 patients received one unit; 23 patients received two units. Median time to ANC >500/µL was 24 days (95% confidence interval 22-28 days), 25 days for one unit and 23 days for two units (P=0.99). At day 100, ANC >500/µL was 88.4 and 91.3% in the one- and two-unit groups (P=0.99), respectively. Three-year EFS was 28.6% and 39.1% in the one- and two-unit groups (P=0.71), respectively. Infusion of two units was associated with a significantly lower relapse risk, 30.4% vs 59.3% (P=0.045). Infused cell doses (TNC, CD3(+), CD34(+) and CD56(+)CD3(neg)) did not impact on engraftment, OS or EFS. Taken together, one-unit UCB transplantation with a threshold cell dose 2.5 × 10(7)/kg recipient weight after RIC is a viable option for adults, although infusion of two units confers a lower relapse incidence.

Regulation of IL-2 expression by transcription factor BACH2 in umbilical cord blood CD4+ T cells.

On activation, umbilical cord blood (UCB) CD4(+) T cells demonstrate reduced expression of tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma), whereas maintaining equivalent interleukin-2 (IL-2) levels, as compared with adult peripheral blood (PB) CD4(+) T cells. Nuclear factor of activated T cells (NFAT1) protein, a transcription factor known to regulate the expression of IL-2, TNF-alpha and IFN-gamma, is reduced in resting and activated UCB CD4(+) T cells. In contrast, expression of Broad-complex-Tramtrack-Bric-a-Brac and Cap'n'collar homology 1 bZip transcription factor 2 (BACH2) was shown by gene array analyses to be increased in UCB CD4(+) T cells and was validated by qRT-PCR. Using chromatin immunoprecipitation, BACH2 was shown binding to the human IL-2 proximal promoter. Knockdown experiments of BACH2 by transient transfection of UCB CD4(+) T cells with BACH2 siRNA resulted in significant reductions in stimulated IL-2 production. Decreased IL-2 gene transcription in UCB CD4(+) T cells transfected with BACH2 siRNA was confirmed by a human IL-2 luciferase assay. In summary, BACH2 maintains IL-2 expression in UCB CD4(+) T cells at levels equivalent to adult PB CD4(+) T cells despite reduced NFAT1 protein expression. Thus, BACH2 expression is necessary to maintain IL-2 production when NFAT1 protein is reduced, potentially impacting UCB graft CD4(+) T-cell allogeneic responses.

In vitro replication models for the hepatitis C virus.

Soon after the discovery of the hepatitis C virus (HCV), attention turned to the development of models whereby replication of the virus could be investigated. Among the HCV replication models developed, the HCV RNA replicon model and the newly discovered infectious cell culture systems have had an immediate impact on the study of HCV replication, and will continue to lead to important advances in our understanding of HCV replication. The aim of this study is to deal with developments in HCV replication models in a chronological order from the early 1990s to the recent infectious HCV cell culture systems.

Viral load change and sequential evolution of entire hepatitis C virus genome in Irish recipients of single source-contaminated anti-D immunoglobulin*.

In hepatitis C virus (HCV) infection, serum viral load is important in the prediction of therapeutic efficacy. However, factors that affect the viral load remain poorly understood. To identify viral genomic elements responsible for the viral load, we investigated samples from a population of Irish women who were iatrogenically infected from a single HCV source by administration of HCV 1b-contaminated anti-D immune globulin between 1977 and 1978 (Kenny-Walsh, N Engl J Med 1999; 340: 1228). About 15 patients were divided into two groups, viral load increasing group (11 patients) and decreasing group (4 patients). Pairs of sera were collected from each patient at interval between 1.1 and 5.8 years. Full-length sequences of HCV genome were determined, and analyzed for changes in each patient. Sliding window analysis showed that the decreasing group had significantly higher mutation rates in a short segment of NS5B region that may affect the activity of RNA-dependent RNA polymerase. By comparing each coding regions, significantly higher mutation numbers were accumulated in NS5A region in the increasing group than the decreasing group (0.92 vs 0.16 nucleotides/site/year, P = 0.021). The mutation in certain positions of the HCV genome may be determinant factors of the viral load in a relatively homogeneous patient population.

Effect of temperature reduction on myotonia in rat skeletal muscles in vitro.

1. The objective of the study was to determine the effect of temperature reduction on the response of rat skeletal muscles to myotonia-inducing agents. 2. A model myotonia was induced in the muscles in vitro, using either the chloride channel blocker anthracene-9-carboxylic acid or chloride-free Krebs solution. This model is similar in its characteristics to the myotonia which occurs in autosomal recessive generalized myotonia congenita in humans. 3. Isometric twitch contractions were recorded in the muscles in Krebs solution before and after the addition of the myotonia-inducing agent. The presence of myotonia was confirmed when the half-relaxation time of the twitch contraction after the addition of the agent was significantly greater than that before its addition. 4. Recordings were made at 37 degrees C, 30 degrees C, 25 degrees C and 15 degrees C. Myotonia developed at 37 degrees C, 30 degrees C and 25 degrees C, but not at 15 degrees C, indicating that at a temperature between 25 degrees C and 15 degrees C, anthracene-9-carboxylic acid-induced myotonia failed to develop. This supports the results obtained in humans suffering from myotonia congenita where myotonic contractions in the adductor pollicis muscle disappeared when the muscle temperature was cooled to 20 degrees C. 5. The myotonia which developed at 37 degrees C could be significantly reduced by exposure to 1 x 10(-4) mol/l ouabain or by elevation of the K+ concentration of the Krebs solution to 7.5 mmol/l. 6. Measurements made using microelectrodes showed that the conditions under which myotonia either did not develop or was significantly reduced, i.e. a temperature of 15 degrees C, exposure to 7.5 mmol/l K+ at 37 degrees C or exposure to 1 x 10(-4) mol/l ouabain at 37 degrees C were each associated with membrane depolarization. The results are discussed in terms of a possible role for depolarization in preventing/reducing the myotonic response.

Sperm acrosin levels in semen: comparison between ACCU-SPERM and Kennedy's methods.

The purpose of this experiment was to evaluate the accuracy, specificity, and sensitivity of a new acrosin activity assay, ACCU-SPERM, and to correlate these results with the original Kennedy method. Thirty-nine specimens (26 patients and 13 donors) of 54 (72%) were found to be in the normal range (> 25 microIU acrosin/10(6) sperm) by the Kennedy method; the other 15 specimens were in either the indeterminate or subfertile range (< 14 microIU). However, according to the ACCU-SPERM method, (normal: 6.6-27 AAI; infertile: < 3.6), 90% of specimens (49 of 54) whose acrosin activity was measured were in the subfertile or infertile range. Similarly, only 28% (4 of 14) of donors in the ACCU-SPERM method were in the normal range in contrast to the 93% (13 of 14) in Kennedy. After calculating the ACCU-SPERM normal range in our laboratory using the linear regression curve between the acrosin values generated by the Kennedy and ACCU-SPERM methods, we again compared results of the two methods. The new normal range of > 1.82 AAI in ACCU-SPERM corresponded to > 25 microIU in the Kennedy method; similarly a value of < 1.35 AAI in ACCU-SPERM corresponded to < 14 microIU in the Kennedy technique. Analysis of the results generated by the two methods revealed a poor correlation with a positive concordance of 51% and a negative concordance of 50% in both assays. These results strongly suggest that the ACCU-SPERM method for measurement of acrosin activity is not a reliable assay.

Relationships between water mass characteristics and estimates of fish population abundance from trawl surveys.

The Canadian Department of Fisheries and Oceans conducts annual bottom trawl surveys to monitor changes in the abundance of the major commercially important groundfish populations. Some of these surveys have been in operation for almost 20 yr. The estimates from these surveys often indicate rapid changes in abundance over time beyond that expected from the population dynamics of the fish. Much of this interannual change has been interpreted as variation, the magnitude of which has often made it difficult to measure anything but the most severe effects of fishing, pollution or any other intervention on the population. Recent studies have shown that some of this variation may be attributed to changes in catchability of fish due to the effects of environmental variables on fish distribution. Annual changes in abundance as estimated from such field surveys may be confounded by changes in catchability due to annual changes in environmental conditions. In this study, trawl catches of age 4 Atlantic cod (Gadus morhua) from surveys conducted during March 1979-1988 were compared with concurrent measurements of bottom salinity, temperature and depth. Large catches of age 4 cod are more likely to occur in water characterized as the intermediate cold layer defined by salinities of 32-33.5 and temperatures<5°C. This relationship also appears to be modified by depth. We further show that internnual changes in the estimated abundance from the surveys were, in a number of cases, coincident with changes in the proportion of the bottom water composed of the intermediate cold water layer. The implications that these patterns may have on interpreting trends in the estimates of abundance from trawl surveys are discussed.

Do Taq-generated RT-PCR products from RNA viruses accurately reflect viral genetic heterogeneity?

Since the first report of genetically heterogeneous, or quasispecies, populations of RNA viruses, the genetic heterogeneity of the RNA genomes of major viral pathogens has been extensively studied. These studies aim to provide insights into the evolutionary pressures that act upon viruses, in order to define windows where anti-viral therapies will be most effective, to take prognostic values from viral genetic distributions at a given time, and to aid the development of novel therapeutic compounds that may tilt viral replication towards information loss. Many methodologies are employed to analyse genetic distributions of a virus in a given sample, but all involve the generation, and subsequent analysis, of the sequence information contained in a reverse-transcription-polymerase chain reaction (RT-PCR) product. Despite the fact that the aim of these RT-PCRs is to obtain sequence information from viral genomes, their application to this task is approached without adequate consideration of this end-goal. The establishment of an RT-PCR for a specific viral target genome generally proceeds in the same fashion as one would apply to establishing a PCR to determine the presence or absence of a specific target sequence in a given sample. However, it is becoming increasingly apparent that RT-PCR products generated by amplification with the ubiquitous thermostable DNA polymerase Taq, coupled with standard cloning and sequencing methodologies, has the potential to yield inaccurate and misleading data as pertains to the information content of populations of RNA viral genomes. This review discusses varying approaches employed to analyse heterogeneous populations of hepatitis C virus RNA genomes.

Purification and characterization of an endonuclease from fruiting caps of basidiomycete Coprinus cinereus.

An endonuclease was purified from the cap tissues of basidiocarp of Coprinus cinereus collected at early meiotic prophase. It has an optimal activity at pH 7.0 and 37 degrees C. It is a cationic enzyme with a molecular mass of 22 kDa by gel filtration, and contains a 12-kDa and a 14-kDa peptide as revealed by SDS gel electrophoresis and Western blot analysis. An antiserum was produced in rabbits against the purified Coprinus endonuclease. The specificity of this antiserum was demonstrated in a dot-blot analysis and, more critically, in an immunoinhibition of endonuclease activity. The Coprinus endonuclease requires Mg2+ and/or Ca2+ as co-factors. Ca2+ is more efficient than Mg2+ while the effect of combining both co-factors is the highest. The Coprinus endonuclease has a substrate preference for single-strand and supercoiled DNA. It gives only single-strand nicks on supercoiled DNA at low enzyme concentration and limited time of incubation. At high enzyme concentration and/or long incubation time, double-strand fragmentation occurred. As is discussed, this endonuclease is believed to be involved in the early phase of meiotic recombination.

Development of the immunoglobulin repertoire.

In this Review we will include studies on the development immunoglobulin repertoire. We will discuss the pattern of V, (D), and J rearrangement in both normal B cells and autoimmune disorders. We will define the role of the recombination signal sequences and the importance of the nucleotide sequence of these highly conserved motifs. Whether deviations from the consensus recombination signal sequence will be tolerated by the recombination mechanism and the importance of the recombination-activating genes are also discussed. We will address the issue of whether pathogenic autoantibodies are generated as part of the normal immune repertoire and the importance of receptor editing as a means by which the immune system deletes autoreactive B cells.